This project studies peripheral blood hematopoietic progenitors (PBHP) as a target for gene therapy of inherited diseases affecting the function of human phagocytic cells, including neutrophils, eosinophils, and monocytes. A related goal of this project is development of novel cellular therapies based on genetically engineering human PBHP and their phagocytic cell progeny to endow them with new properties to augment host defense against chronic infections with intracellular pathogens including tuberculosis and other mycobacteria infections. We have defined conditions for optimum harvest, purification and culture of the primitive human hematopoietic cells with a CD34 surface antigen phenotype. In a clinical study we determined the optimum dosing of subcutaneous granulocyte colony stimulating factor (G-CSF) to recruit primitive CD34 progenitor cells from bone marrow to peripheral blood. We showed that mobilization is impaired in patients with chronic granulomatous disease and ADA-deficient SCID. We have studied optimum conditions for growth of CD34+ cells in culture, developing conditions leading to predominantly neutrophils, eosinophils or monocyte/macrophages. This will be important for studies aimed at engineering new characteristics into specific end stage phagocytes by gene transfer into progenitors. Using conditions for monocyte differentiation we introduced a gene for interferon gamma (IFN-gamma) into these cells by targeting the CD4+ progenitors, finding that this resulted in augmentation of oxidase activity and an increase in IgG Fc receptors. In other studies, the gene for nitric oxide synthase (NOS) was transferred into the human erythroleukemia cells line, finding that the constituitive nitric oxide production resulted in changes in post-translational regulation of several genes related to iron metabolism and hemoglobin production. This may provide insight into the mechanisms responsible for anemia of chronic infection. In studies of transplantation in mice, we have demonstrated that a combination of growth factor administration followed by low dose radiation is an effective method for enhancing engraftment of transfused congenic progenitor cells. This has important implications for engraftment following gene therapy in humans targeting autologous CD34+ cells.